The invention relates to the field of cell type-specific gene expression. Dopamine β-hydroxylase (DBH) is a hallmark protein of noradrenergic neurons because noradrenaline is synthesized by this enzyme. The highly restricted pattern of DBH expression in the nervous system predicts that this gene is subject to neuron-specific as well as to cell type-specific control mechanisms. Transgenic mice experiments have shown that 5.8 or 4 kb of the 5′ flanking sequences of the human DBH gene can drive expression of the reporter gene in neurons of the locus coeruleus as well as other noradrenergic neurons and adrenal chromaffin cells, albeit with some ectopic expression. More recently, comparison of reporter gene expression in transgenic animals generated by using DBH 5′ flanking regions of different lengths indicated that the upstream region between −1.1 and −0.6 kb is necessary for expression in adult and fetal noradrenergic neurons. Using cell culture systems, we and others have demonstrated that the 5′ upstream region of the DBH gene can drive reporter gene expression in a cell-specific manner.
Deletional and site-directed mutational analyses have indicated that as little as 486 bp of the upstream sequence of the human DBH gene can direct expression of a reporter gene in a cell type-specific manner. In the 486 bp region of the human DBH gene, the distal part spanning −486 to −263 bp appears to have a cell-specific silencer function that contributed to suppression of the promoter activity in nonneuronal cells. Transient transfection assays identified the proximal part spanning −262 to +1 bp as sufficient and essential for the high-level DBH promoter activity in DBH-positive cells. In this 262 bp proximal area, four protein-binding regions (domains I to IV) have been identified by DNase I footprinting analysis. A cAMP response element (CRE), 5′-TGACGTCC-3′ (SEQ ID NO: 3), with a single base deviation from the consensus octamer motif, 5′-TGACGTCA-3′ (SEQ ID NO: 4), was shown to be critical for both the basal and cAMP-inducible transcription in DBH-expressing cell lines. This CRE is included in a composite enhancer domain structure located at −185 to −150 bp, designated domain IV, which contains several additional cis-elements such as AP1, YY1, and two core motifs of homeodomain (HD) binding sites. Site-directed mutagenesis of each sequence motif has revealed that the CRE is essential for basal promoter activity in every cell line, YY1 is multifunctional, and the AP1-like motif may be transcriptionally inactive.
The murine paired-like HD protein, Phox2a, is selectively expressed in noradrenergic cells and is critical for development of several noradrenergic neuron populations, including the locus coeruleus. The forced expression of Phox2a robustly activates DBH promoter activity, strongly suggesting a mechanism for noradrenergic-specific promoter function. Moreover, Phox2b, which contains an HD identical to that of Phox2a, has been identified and shown to be widely coexpressed with Phox2a in both the central and peripheral nervous system. Cotransfection assays showed that Phox2a and Phox2b transactivate the DBH promoter activity with a comparable efficiency.